Recent experimental evidence has implicated membrane and cytoskeletal elements as modifiers of adenylate cyclase (AC) activity in rat cerebral cortex synaptic membranes. This study will attempt to explore molecular interaction between tubulin and the AC regulatory subunit (G-unit). Furthermore, we wish to determine how these interactions might participate in the regulation of AC, especially in nerve cells. G-unit will be covalently labelled with a photoaffinity probe (radioactive or flourescent) and isolated through affinity chromatography in a tubulin column. G-unit will also be electrophoretically separated and hybridized with tubulin. The nature of G-unit tubulin interaction will be explored through co-sedimentation studies in sucrose density gradients and gel filtration media. Preliminary indications are that this putative G-unit tubulin interaction is specific to cells of neuronal origin. This will be explored further and, if G-unit from nerve cells proves distinct from other tissues, its purification wil be attempted utilizing a tubulin affinity column to distinguish the brain specific form. As Ca2+/calmodulin activated AC appears distinct to cells of neuronal origin, the interplay between Ca2+/calmodulin activation of AC and activation of the enzyme by colchicine or vinblastine will be examined. Sequential addition and reconstitution experiments, will be employed to determine the mechanisms of cytoskeletal involvement in the AC activation process. Similar experiment will be performed to distinguish stimulatory from inhibitory G-unit with regard to their interaction with tubulin. Binding and release of guanyl nucleotides will be followed usng a fluorescent GTP probe to determine how the structure and function of the G-unit is altered by nucleotide binding. We have previously noted changes in adenylate cyclase coupling (at the level of the G-unit) after long-term antidepressant treatment. Consequently, understanding possible participation of membrane and cytoskeletal elements in the regulation of brain AC might lead to the designing of pharmacological strateges to treat various psychiatric, neurologic and metabolic disorders.